Extended Data Fig. 7: The EAAT1 open-channel conformation conducts Cl−.
From: Glutamate transporters have a chloride channel with two hydrophobic gates
a, l-[3H]glutamate uptake into oocytes expressing cysteine-less EAAT1 and double cysteine transporter mutants in control conditions (grey), and after pre-incubation with DTT (cyan) or copper phenanthroline (orange). Number of cells (n) used for each condition is indicated in each graph and all measurements presented were taken across at least two batches of oocytes. b–e, l-Glutamate elicited current–voltage relationships for cysteine-less E1 (b), E1-XL1 (c), E1-XL2 (d) and E1-XL3 (e) monitored under the same conditions as a. f, g, To confirm that crosslinks E1-XL1, E1-XL2 and E1-XL3 were occurring within an individual protomer, rather than between protomers of the trimeric complex, oocytes expressing single cysteine residues that make up E1-XL1 (K300C and W473C), E1-XL2 (L244C and G439C), and E1-XL3 (K300C and A470C) either alone or co-injected into an individual oocyte were also examined using the same approaches as in a, b–e. Data are mean ± s.e.m. h, EAAT1 (PBD: 5LLU) highlighting residues forming the extracellular and intracellular hydrophobic gates. The scaffold domain is shown in grey and the transport domain in gold. The Cα atoms of the two introduced cysteine residues are shown as spheres (L244 in red and G439 in blue). i, Membrane reversal potentials (Erev) measured in oocytes expressing wild-type (n = 6) and mutant (n = 5) EAAT1 transporters. Each data point (white circle) represents a response from a single cell. The black bar represents mean ± s.e.m. Significance was determined using one-way ANOVA with Bonferroni post hoc analysis for multiple comparisons performed using GraphPad Prism 8; exact P values are provided. j, Schematic of the substrate-transport cycle. A single protomer is shown with the scaffold domain in salmon, the transport domain in blue and the substrate in black.
